As used herein, the term “stem cell” generally refers to cells that have excellent self-renewal potential while maintaining an undifferentiated state and are capable of differentiating in a tissue-specific manner so as to have certain functions and shapes under certain environments and conditions. Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells, are capable of self-renewal under suitable in vitro culture conditions and have a pluripotent ability to differentiate into all types of cells of the body. Due to such characteristics, the results of studies on these pluripotent stem cells have been applied not only for the understanding of biological basic knowledge, including the development, differentiation and growth of organisms, but also for the development of cell therapy agents for fundamental treatment of various diseases and the development of new drugs. While efforts have been increasingly made to develop practically applicable technology based on human pluripotent stem cells in various fields, there are still problems to be solved in terms of efficiency, safety and economy in a process for the production and proliferative culture of human pluripotent stem cells. Specifically, it is required to develop a technology for producing large amounts of undifferentiated and differentiated stem cells, which can satisfy the demand for the stem cells at any time. Particularly, for the development of cell therapy agents, it is necessary to ensure cell culture technology, which has excellent performance, can provide clinically applicable cells and is highly efficient.
Generally, undifferentiated human pluripotent stem cells can be continuously cultured by co-culturing with feeder cells such as mouse embryonic fibroblasts (MEFs) or in feeder-free conditions using conditioned media (CM) obtained from cultures of MEFs or chemically defined media. However, co-culture with animal feeder cells or the use of conditioned media from animal feeder cells involves the risk of transmitting one or more infectious agents such as viruses to human pluripotent stem cells. For this reason, in recent years, continued efforts have been made to develop chemically defined media containing known components such as low-molecular-weight compounds, peptides or the like without containing animal feeder cells or sera and apply these chemically defined media for the production and culture of human pluripotent stem cells. These chemically defined media have significantly contributed to the growth of the stem cell market.
Embryonic stem cells derived from the inner cell mass of frozen-thawed embryos are extracted from frozen-thawed embryos to be discarded, and thus pose no legal problems. However, because these cells are extracted from embryos, these cells pose ethical and religious issues from the viewpoint of life's destruction. In addition, because these cells are derived from limited embryos, transplant rejection cannot be avoided due to the lack of immune compatibility between individuals. As an alternative to overcome such problems, a method of producing induced pluripotent stem cells having characteristics almost similar to those of embryonic stem cells from adult stem cells using reprogramming factors was recently successfully developed (Cell 126, 663-676, 2006; Cell 131, 861-872, 2007; Nature 441, 1061-1067, 2006; Nature 451, 141-146, 2008). Due to the development of this method, the expectation of development of practically applicable stem cell technology based on pluripotent stem cells is increasing. Particularly, induced pluripotent stem cells do not require embryos for the production thereof, the use of the patient's own extracted stem cells does not pose the problem of immune rejection, and thus induced pluripotent stem cells are technically highly useful. However, in order to allow the current technology to enter the stage of practical stage, it is necessarily required to develop technology capable of replacing the use of virus in order to overcome the problems of the current technology, including low efficiency of reprogramming and low clinical safety.
As methods for improving the efficiency of reprogramming, examples of success in increasing the efficiency of reprogramming by the control of extracellular environments or the use of additives such as low-molecular-weight compounds have been reported. In addition, it was reported that the efficiency of reprogramming of somatic cells is effectively increased in a hypoxic condition similar to the environment of embryonic stem cells (Cell Stem Cell, 5: 237-241, 2009). Also, Dr. Ding's team (Shi et al., Cell Stem Cell, 2008) reported that low-molecular-weight compounds such as BIX-01294 (G9a histone methyltransferase inhibitor) and BayK8644 (L-type calcium channel agonist), RG108 (DNA methyltransferase inhibitor) are effective in increasing the efficiency of reprogramming, and Dr. Melton's team (Huangfu et al., Nat Biotechnol, 2008) reported that low-molecular-weight compounds such as VPA (histone deacetylase inhibitor), TSA (histone deacetylase inhibitor) and SAHA (histone deacetylase inhibitor are effective in increasing the efficiency of reprogramming. With respect to the development of alternatives to the use of virus, the following study results were reported: 1) transient expression technology utilizing a single nonviral polycistronic vector (Gonzalez et al, PNAS USA, 2009; Chang et al, Stem cells, 2009); 2) adenoviral transfection technology (Stadtfeld et al, Science 2008); 3) establishment of iPSC using a single nonviral polycistronic vector through the development of a Cre/loxP recombinant expression control system (Soldner et al, Cell, 2009), and removal of a reprogramming cassette by Cre transfection (Kaji et al, Nature, 2009), 4) a piggyback (PB) transposon system (Woltjen et al, Nature, 2009; Kaji et al, Nature, 2009), and 5) nonintegrating episomal vectors (Yu et al, Science, 2009). However, the possibility of genetic abnormalities and tumorigenesis still exists.
Accordingly, the present inventors have conducted extensive studies to discover not only low-molecular-weight compounds capable of improving the technology for the maintenance and culture of undifferentiated human pluripotent stem cells, but also low-molecular-weight compounds capable of improving the reprogramming technology of producing human pluripotent stem cells from somatic cells. As a result, the present inventors have discovered the novel low-molecular-weight compound RSC-133 and have verified that the use of a medium composition containing RSC-133 significantly increases the efficiency of reprogramming for producing human pluripotent stem cells and significantly improves culture conditions for maintaining and proliferating human pluripotent stem cells in an undifferentiated state, thereby completing the present invention.